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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2017, Vol. 07 ›› Issue (02) : 81 -86,f3-f8. doi: 10.3877/cma.j.issn.2095-1221.2017.02.004

所属专题： 文献；

论著

两种人脐带间充质干细胞源外泌体分离方法的比较
刘高米洋1, 潘兴华2, 和法莲3, 陆容4, 刘菊芬2, 何洁2, 王红阳5,†()   
  1. 1. 200438　上海，第二军医大学东方肝胆外科医院国际合作生物信号转导研究中心；650032　昆明，成都军区昆明总医院细胞生物治疗中心　干细胞与免疫细胞生物医药技术国家地方联合实验室　云南省细胞治疗技术转化医学重点实验室
    2. 650032　昆明，成都军区昆明总医院细胞生物治疗中心　干细胞与免疫细胞生物医药技术国家地方联合实验室　云南省细胞治疗技术转化医学重点实验室
    3. 650032　昆明医科大学成都军区昆明总医院临床学院
    4. 200062　上海市儿童医院儿科
    5. 200438　上海，第二军医大学东方肝胆外科医院国际合作生物信号转导研究中心
  • 收稿日期:2016-08-02 出版日期:2017-04-01
  • 通信作者: 王红阳
  • 基金资助:
    国家自然科学基金(81170316)

Comparisons of two isolation methods for exosomes from human umbilical cord mesenchymal stem cells

Miyang Liu-Gao1, Xinghu Pan2, Falian He3, Rong Lu4, Jufen Liu2, Jie He2, Hongyang Wang5,†()   

  1. 1. International Co-operation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China; Cell Biological Therapy Center of Kunming General Hospital, Chengdu Military Region of Chinese People's Liberation Army, Cell Biological Medicine integrated Engineering Laboratory of State and Region of Yunnan Province, Kunming 650032, China
    2. Cell Biological Therapy Center of Kunming General Hospital, Chengdu Military Region of Chinese People's Liberation Army, Cell Biological Medicine integrated Engineering Laboratory of State and Region of Yunnan Province, Kunming 650032, China
    3. Kunming General Hospital Clinical Collage of Kunming Medical University, Kunming 650032, China
    4. Department of Pediatric, Shanghai Children's Hospital, Shanghai 200062, China
    5. International Co-operation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Institute, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China
  • Received:2016-08-02 Published:2017-04-01
  • Corresponding author: Hongyang Wang
  • About author:
    Corresponding author: Wang Hongyang, Email:
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引用本文：

刘高米洋, 潘兴华, 和法莲, 陆容, 刘菊芬, 何洁, 王红阳. 两种人脐带间充质干细胞源外泌体分离方法的比较[J]. 中华细胞与干细胞杂志(电子版), 2017, 07(02): 81-86,f3-f8.

Miyang Liu-Gao, Xinghu Pan, Falian He, Rong Lu, Jufen Liu, Jie He, Hongyang Wang. Comparisons of two isolation methods for exosomes from human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2017, 07(02): 81-86,f3-f8.

目的

探索人脐带间充质干细胞（hUMSCs）来源的外泌体的最佳分离条件并对其进行生物学鉴定。

方法

无血清培养法培养hUMSCs，培养上清中收集外泌体，超高速离心法（ult-exo）和沉淀法分离外泌体（pri-exo），Western Blot检测外泌体标志性蛋白CD63、HSP70、HSP90和阴性对照蛋白TAPA1，透射电镜观察分离所得外泌体生物学形态，高清晰质谱分析外泌体包裹蛋白种类。

结果

无血清培养法能够在不改变细胞生物学形态的条件下获取hUMSCs源外泌体，透射电镜下可见超高速离心可以获得具有典型双层膜结构的外泌体，大小在100 nm左右，而沉淀法分离所得产物不具有典型的外泌体形态表征，大小在30 nm左右；Western Blot结果表明超高速离心法所得外泌体CD63、HSP70、HSP90均呈现阳性，阴性对照蛋白TAPA1呈现阴性，沉淀法只能检测到HSP90；高清晰质谱检测出超高速离心法分离所得外泌体有169种蛋白，沉淀法有102种，共有蛋白79种。

结论

超高速离心法和沉淀法所得外泌体在蛋白标志物、生物学形态和内含蛋白种类均有差别，本实验结果提示分离hUMSCs源外泌体超高速离心法优于沉淀法。

关键词: 脐带, 间质干细胞, 外泌体, 分离, 鉴定
Objective

To explore the best method of separating exosomes secreted by human umbilical cord mesenchymal stem cells (huMSCS) .

Methods

Exosomes were collected from huMSCS in the serum-free medium culture system by either ultracentrifugation or precipitation methods. Western Blot was used to detect TAPA1, CD63, HSP70, and HSP90 of exosomes. Tansmission electron microscope was used to observe exosome morphology, and proteome of exosomes isolated via the two methods was compared.

Results

Exosomes isolated via ultracentrifigation were around 100 nm in diameter under transmission electron microscopy and were positive for CD63, HSP70, and HSP90, while negative for TAPA1; exosomes isolated via 101 Kit were around 30 nm in diameter and were positive only for HSP90. High-resolution mass spectrometry (HRMS) revealed 169 proteins in exosomes isolated via ultracentrifigation while only 102 proteins were identified in exosomes isolated via 101 Kit. Comparative analysis dates from HRMS indicated that there were 79 proteins shared by exosomes isolated by the two methods.

Conclusion

Exosomes isolated by ultracentrifugation and precipitation are different in biomarkers and proteome profile, and ultracentrifugation method is superior to precipitation method.

Key words: Umbilical Cord, Mesenchymal Stem Cells, Exosomes, Isolation, Identification
图1 倒置显微镜下观察人脐带间充质干细胞的生长形态（×40）
图2 电镜下观察两种方法得到的外泌体形态（×67 000）
图3 银染检测外泌体蛋白丰度
图4 Western Blot检测外泌体标志
图5 高清晰质谱分析外泌体蛋白组
表1 ult-exo蛋白列表
UNIPROT_ACCESSION NAME
P27824 calnexin(CANX) Homo sapiens
P04217 alpha-1-B glycoprotein(A1BG)
P68104 eukaryotic translation elongation factor 1 alpha 1(EEF1A1)
P61626 lysozyme(LYZ)
P22735 transglutaminase 1(TGM1)
P22792 carboxypeptidase N subunit 2(CPN2)
P07585 decorin(DCN)
P01011 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3(SERPINA3)
Q5T749 keratinocyte proline rich protein(KPRP)
P61769 beta-2-microglobulin(B2M)
P00450 ceruloplasmin (ferroxidase)(CP)
P29966 myristoylated alanine rich protein kinase C substrate(MARCKS)
P25311 alpha-2-glycoprotein 1, zinc-binding(AZGP1)
P0CG05 immunoglobulin lambda constant 2 (Kern-Oz- marker)(IGLC2)
P12883 myosin, heavy chain 7, cardiac muscle, beta(MYH7)
Q16610 extracellular matrix protein 1(ECM1)
P07996 thrombospondin 1(THBS1)
P27482 calmodulin like 3(CALML3)
P04792 heat shock protein family B (small) member 1(HSPB1)
Q9H1J7 wingless-type MMTV integration site family member 5B(WNT5B)
P01033 TIMP metallopeptidase inhibitor 1(TIMP1)
P35542 serum amyloid A4, constitutive(SAA4)
P0C0L5 complement component 4B (Chido blood group), copy 2(C4B_2)
P12235 solute carrier family 25 member 4(SLC25A4)
Q8N1N4 keratin 78, type II(KRT78)
Q66K66 transmembrane protein 198(TMEM198)
Q5D862 filaggrin family member 2(FLG2)
P11021 heat shock protein family A (Hsp70) member 5(HSPA5)
P02788 lactotransferrin(LTF)
Q86YZ3 hornerin(HRNR)
P31151 S100 calcium binding protein A7(S100A7)
P51884 lumican(LUM)
P07355 annexin A2(ANXA2)
P05109 S100 calcium binding protein A8(S100A8)
P02654 apolipoprotein C-I(APOC1)
P06307 cholecystokinin(CCK)
P02652 apolipoprotein A-II(APOA2)
P21741 midkine (neurite growth-promoting factor 2)(MDK)
P68871 hemoglobin subunit beta(HBB)
P23490 loricrin(LOR)
P05090 apolipoprotein D(APOD)
P02655 apolipoprotein C-II(APOC2)
P12109 collagen type VI alpha 1(COL6A1)
P68371 tubulin beta 4B class IVb(TUBB4B)
P15924 desmoplakin(DSP)
P12110 collagen type VI alpha 2(COL6A2)
B4DZ36 attractin(ATRN)
P12111 collagen type VI alpha 3(COL6A3)
Q02413 desmoglein 1(DSG1)
P02647 apolipoprotein A-I(APOA1)
P02458 collagen type II alpha 1(COL2A1)
P02461 collagen type III alpha 1(COL3A1)
P20929 nebulin(NEB)
Q08554 desmocollin 1(DSC1)
P31944 caspase 14(CASP14)
P05543 serpin peptidase inhibitor, clade A , member 7(SERPINA7)
P62807 histone cluster 1, H2bd(HIST1H2BD)
P02753 retinol binding protein 4(RBP4)
Q16270 insulin like growth factor binding protein 7(IGFBP7)
P05089 arginase 1(ARG1)
P06276 butyrylcholinesterase(BCHE)
P35611 adducin 1(ADD1)
P69905 hemoglobin subunit alpha 1(HBA1)
P27487 dipeptidyl peptidase 4(DPP4)
P25788 proteasome subunit alpha 3(PSMA3)
P01834 immunoglobulin kappa constant(IGKC)
P00738 haptoglobin(HP)
Q6KB66 keratin 80, type II(KRT80)
P62987 ubiquitin A-52 residue ribosomal protein fusion product 1(UBA52)
P43251 biotinidase(BTD)
P14209 CD99 molecule(CD99)
P10599 thioredoxin(TXN)
P09486 secreted protein acidic and cysteine rich(SPARC)
P08697 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2(SERPINF2)
P20930 filaggrin(FLG)
Q9Y3U8 ribosomal protein L36(RPL36)
P13667 protein disulfide isomerase family A member 4(PDIA4)
O95445 apolipoprotein M(APOM)
P05556 integrin subunit beta 1(ITGB1)
P02763 orosomucoid 1(ORM1)
P02766 transthyretin(TTR)
P04406 glyceraldehyde-3-phosphate dehydrogenase(GAPDH)
P19652 orosomucoid 2(ORM2)
P01040 cystatin A(CSTA)
Q96C01 family with sequence similarity 136 member A(FAM136A)
P08571 CD14 molecule(CD14)
P02751 fibronectin 1(FN1)
Q08380 lectin, galactoside-binding, soluble, 3 binding protein(LGALS3BP)
P23396 ribosomal protein S3(RPS3)
P80108 glycosylphosphatidylinositol specific phospholipase D1(GPLD1)
P62899 ribosomal protein L31(RPL31)
Q13835 plakophilin 1(PKP1)
P62805 histone cluster 4, H4(HIST4H4)
Q6EMK4 vasorin(VASN)
P62424 ribosomal protein L7a(RPL7A)
P43652 afamin(AFM)
P22531 small proline rich protein 2E(SPRR2E)
P14618 pyruvate kinase, muscle(PKM)
P07339 cathepsin D(CTSD)
P81605 dermcidin(DCD)
Q5T750 chromosome 1 open reading frame 68(C1orf68)
P08123 collagen type I alpha 2(COL1A2)
P02452 collagen type I alpha 1(COL1A1)
Q14847 LIM and SH3 protein 1(LASP1)
P22528 small proline rich protein 1B(SPRR1B)
Q7Z7G0 ABI family member 3 binding protein(ABI3BP)
P46779 ribosomal protein L28(RPL28)
P13637 ATPase Na+/K+ transporting subunit alpha 3(ATP1A3)
Q14677 clathrin interactor 1(CLINT1)
P08727 keratin 19, type I(KRT19)
P02765 alpha-2-HS-glycoprotein(AHSG)
Q96PD5 peptidoglycan recognition protein 2(PGLYRP2)
Q15758 solute carrier family 1 member 5(SLC1A5)
P27090 transforming growth factor, beta 2(Tgfb2)
Q04695 keratin 17, type I(KRT17)
Q96KN2 carnosine dipeptidase 1 (metallopeptidase M20 family)(CNDP1)
P01308 insulin(INS)
P01008 serpin peptidase inhibitor, clade C (antithrombin), member 1(SERPINC1)
P02768 albumin(ALB)
Q9NY65 enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase(EHHADH)
P04259 keratin 6B, type II(KRT6B)
P13647 keratin 5, type II(KRT5)
P10909 clusterin(CLU)
P19013 keratin 4, type II(KRT4)
P04264 keratin 1, type II(KRT1)
P35908 keratin 2, type II(KRT2)
P26022 pentraxin 3(PTX3)
P48509 CD151 molecule (Raph blood group)(CD151)
P51164 ATPase H+/K+ transporting beta subunit(ATP4B)
Q01469 fatty acid binding protein 5(FABP5)
P04075 aldolase, fructose-bisphosphate A(ALDOA)
P01009 serpin peptidase inhibitor, clade A , member 1(SERPINA1)
P09919 colony stimulating factor 3(CSF3)
P08779 keratin 16, type I(KRT16)
Q9UNN8 protein C receptor(PROCR)
Q96DR8 mucin like 1(MUCL1)
P00491 purine nucleoside phosphorylase(PNP)
P07093 serpin peptidase inhibitor, clade E , member 2(SERPINE2)
Q9HCY8 S100 calcium binding protein A14(S100A14)
P67936 tropomyosin 4(TPM4)
P16070 CD44 molecule (Indian blood group)(CD44)
P62249 ribosomal protein S16(RPS16)
P08962 CD63 molecule(CD63)
P13645 keratin 10, type I(KRT10)
P02786 transferrin receptor(TFRC)
P35527 keratin 9, type I(KRT9)
P02774 group-specific component (vitamin D binding protein)(GC)
P05787 keratin 8, type II(KRT8)
Q00610 clathrin heavy chain(CLTC)
表2 pri-exo蛋白列表
UNIPROT_ACCESSION Name
P43251 biotinidase(BTD)
P10599 thioredoxin(TXN
P04217 alpha-1-B glycoprotein(A1BG)
P09486 secreted protein acidic and cysteine rich(SPARC)
P68104 eukaryotic translation elongation factor 1 alpha 1(EEF1A1)
P08697 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2(SERPINF2)
P07585 decorin(DCN)
P20930 filaggrin(FLG)
P01011 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3(SERPINA3)
Q5T749 keratinocyte proline rich protein(KPRP)
P02763 orosomucoid 1(ORM1)
P02766 transthyretin(TTR)
P19652 orosomucoid 2(ORM2)
P0CG05 immunoglobulin lambda constant 2 (Kern-Oz- marker)(IGLC2)
P25311 alpha-2-glycoprotein 1, zinc-binding(AZGP1)
P27482 calmodulin like 3(CALML3)
Q08380 lectin, galactoside-binding, soluble, 3 binding protein(LGALS3BP)
P07996 thrombospondin 1(THBS1)
P22531 small proline rich protein 2E(SPRR2E)
P43652 afamin(AFM)
P01033 TIMP metallopeptidase inhibitor 1(TIMP1)
P0C0L5 complement component 4B (Chido blood group), copy 2(C4B_2)
P81605 dermcidin(DCD)
Q8N1N4 keratin 78, type II(KRT78)
Q66K66 transmembrane protein 198(TMEM198)
P08123 collagen type I alpha 2(COL1A2) Homo sapiens
Q5D862 filaggrin family member 2(FLG2)
P02452 collagen type I alpha 1(COL1A1)
P02788 lactotransferrin(LTF)
Q86YZ3 hornerin(HRNR)
P31151 S100 calcium binding protein A7(S100A7)
P51884 lumican(LUM)
P05109 S100 calcium binding protein A8(S100A8)
P06307 cholecystokinin(CCK)
P08727 keratin 19, type I(KRT19)
P02652 apolipoprotein A-II(APOA2)
P02765 alpha-2-HS-glycoprotein(AHSG)
P68871 hemoglobin subunit beta(HBB)
Q96PD5 peptidoglycan recognition protein 2(PGLYRP2)
P27090 transforming growth factor, beta 2(Tgfb2)
P05090 apolipoprotein D(APOD)
Q04695 keratin 17, type I(KRT17)
P01308 insulin(INS)
P15924 desmoplakin(DSP)
B4DZ36 attractin(ATRN)
P02768 albumin(ALB)
P02458 collagen type II alpha 1(COL2A1)
P02461 collagen type III alpha 1(COL3A1)
P04259 keratin 6B, type II(KRT6B)
P05543 serpin peptidase inhibitor, clade A , member 7(SERPINA7)
P31944 caspase 14(CASP14)
P02753 retinol binding protein 4(RBP4)
P13647 keratin 5, type II(KRT5)
Q16270 insulin like growth factor binding protein 7(IGFBP7)
P19013 keratin 4, type II(KRT4)
P04264 keratin 1, type II(KRT1
P35908 keratin 2, type II(KRT2)
P26022 pentraxin 3(PTX3)
P05089 arginase 1(ARG1)
P09919 colony stimulating factor 3(CSF3)
P35611 adducin 1(ADD1)
P08779 keratin 16, type I(KRT16)
P69905 hemoglobin subunit alpha 1(HBA1)
Q96DR8 mucin like 1(MUCL1)
P01834 immunoglobulin kappa constant(IGKC)
P67936 tropomyosin 4(TPM4)
Q6KB66 keratin 80, type II(KRT80)
P00738 haptoglobin(HP)
P13645 keratin 10, type I(KRT10)
P02786 transferrin receptor(TFRC)
P35527 keratin 9, type I(KRT9)
P02774 group-specific component (vitamin D binding protein)(GC)
P05787 keratin 8, type II(KRT8)
P62987 ubiquitin A-52 residue ribosomal protein fusion product 1(UBA52)
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